Defined sequence segments of the small heat shock proteins HSP25 and alphaB-crystallin inhibit actin polymerization.

The interaction of small heat shock proteins (sHSPs) with the actin cytoskeleton has been described and some members of this family, e.g. chicken and murine HSP25 (HSP27), inhibit the polymerization of actin in vitro. To analyse the molecular basis of this interaction, we synthesized a set of overlapping peptides covering the complete sequence of murine HSP25 and tested the effect of these peptides on actin polymerization in vitro by fluorescence spectroscopy and electron microscopy. Two peptides comprising the sequences W43 to R57 (peptide 6) and I92 to N106 (peptide 11) of HSP25 were found to be potent inhibitors of actin polymerization. Phosphorylation of N-terminally extended peptide 11 at serine residues known to be phosphorylated in vivo resulted in decline of their inhibitory activity. Interestingly, peptides derived from the homologous peptide 11 sequence of murine alphaB-crystallin showed the same behaviour. The results suggest that both HSP25 and alphaB-crystallin have the potential to inhibit actin polymerization and that this activity is regulated by phosphorylation.

Induction of the small stress protein, hsp25, in Ehrlich ascites carcinoma cells by anticancer drugs.

Treatment of in vitro cultured Ehrlich ascites carcinoma cells with cisplatin, daunomycin, doxorubicin, cytosine arabinoside, 3'-fluorodeoxythymidine, colchicine and vincristine in cytostatically effective concentrations results in significantly increased levels of the small stress protein, hsp25, as analyzed by immunoblotting. However, no induction of hsp25 could be detected after treatment of the tumour cells with 5-fluorouracil, aminopterin, amethopterin, mithramycin and cyclophosphamide. None of these cytostatic drugs induces hsp70.

Translational regulation of the mammalian growth-related protein P23: involvement of eIF-4E.

Synthesis of the mammalian growth-related protein P23 is rapidly induced after serum stimulation of mouse fibroblasts and Ehrlich ascites tumour cells. This induction occurs at the translational level. Growth-induction leads also to an increase in phosphorylation of the rate-limiting initiation factor eIF-4E. Here, we present the following evidence indicating the involvement of eIF-4E in the regulation of P23 synthesis: 1) P23 synthesis is induced by the same mitogenic stimuli which lead to enhanced eIF-4E phosphorylation. 2) Upon heat shock treatment of Ehrlich ascites cells (which results in immediate dephosphorylation and concomitant inactivation of eIF-4E), P23 synthesis is rapidly shut off. 3) In control NIH 3T3 cells, P23 synthesis is readily induced by growth stimulation. This response is strongly diminished in cells overexpressing eIF-4E, and the basal level of P23 synthesis is elevated in these cells. Overexpression of a nonfunctional mutant of eIF-4E diminishes the basal level of P23 synthesis as well as the serum-response of the cells with respect to P23 induction. 4) Cells transformed by overexpression of the ras or src genes in which eIF-4E is highly phosphorylated do not show any inducibility of P23 synthesis. 5) HeLa cells expressing antisense RNA of eIF-4E, have reduced levels of eIF-4E/F and show reduced rates of growth and protein synthesis. In these cells the total amount of P23 protein is about 50% compared with control cells. The results suggest that P23 is one of the gene products, the synthesis of which is regulated by eIF-4E activity.

Over-expression of the small heat-shock protein, hsp25, inhibits growth of Ehrlich ascites tumor cells.

hsp25 is a small, growth-related, mammalian stress protein which is highly accumulated in the stationary phase of Ehrlich ascites tumor in vivo. Ehrlich ascites cells cultivated in vitro under conditions of continuous exponential growth express hsp25 only at a low level. These cells were stably transfected with an eukaryotic expression vector carrying the coding sequence of the small heat-shock protein, hsp25, under control of the murine metallothionein promoter. The resulting cell lines (EAT II6 and EAT II8) exhibit constitutive over-expression of the small heat-shock protein, hsp25, which can be further increased by induction with cadmium. Both cell lines show increased thermoresistance. The in vitro proliferation rate of the transfected cell lines EAT II6 and EAT II8 is significantly decreased depending on the degree of cadmium-regulated over-expression of hsp25. Furthermore, a significant delay in Ehrlich ascites tumor growth in mice using the hsp25 over-expressing cells for primary inoculation could be demonstrated.

The small heat shock protein hsp25 is accumulated in P19 embryonal carcinoma cells and embryonic stem cells of line BLC6 during differentiation.

Murine embryonal carcinoma and embryonic stem cell lines were investigated with regard to the occurrence of the small heat shock protein hsp25 during cell growth and differentiation. In the embryonal carcinoma cell line F9 considerable constitutive levels of hsp25 were observed which could be slightly increased by treatment with retinoic acid. No hsp25 was found, however, in the embryonal carcinoma cell line PCC4. When analyzing the pluripotent embryonal carcinoma cell line P19 and the pluripotent embryonic stem cell line BLC6, both characterized by high differentiation capacity, no hsp25 was observed under cell culture conditions maintaining the undifferentiated state. Induction of differentiation caused by prolonged cell culture, retinoic acid treatment, or embryoid body formation, however, resulted in an increase of the level of hsp25. The finding that hsp25 is accumulated in a differentiation-dependent manner suggests that this protein is associated with processes involved in differentiation. Therefore, hsp25 can be regarded as a marker of differentiation in the investigated embryonal carcinoma cell line P19 and the embryonic stem cell line BLC6.

Cell-free phosphorylation of the murine small heat-shock protein hsp25 by an endogenous kinase from Ehrlich ascites tumor cells.

The small heat-shock protein hsp25 of the Ehrlich ascites tumor exists in one non-phosphorylated (hsp25/1) and two phosphorylated (hsp25/2, hsp25/3) isoforms. In stationary phase tumor cells, a protein kinase activity was detected which phosphorylates hsp25/1, resulting in the formation of several phosphorylated hsp25 isoforms, including those occurring naturally in the tumor. Cell-free phosphorylation of hsp25 required Mg2+ and ATP and was independent of Ca2+, phosphatidylserine, cAMP and cGMP. Polymyxin B inhibited, specifically, hsp25 phosphorylation, whereas trifluoperazine, staurosporine and the protein inhibitor of protein kinase A had no effect. In its properties, the hsp25 phosphorylating kinase differs from other common kinases such as protein kinases A and C, calcium/calmodulin-dependent kinases, and the ribosomal protein S6 kinase.

Cisplatin induces the small heat shock protein hsp25 and thermotolerance in Ehrlich ascites tumor cells.

Exposure of Ehrlich ascites tumor (EAT) cells to the anticancer drug cisplatin results in an elevated abundance of three isoforms of the small heat shock protein hsp25 without inducing the general stress response as commonly observed after heat shock. The most effective cisplatin concentration (2.5 microM) is also most efficient in arresting cells in S phase suggesting a relationship between hsp25 expression and cell cycle events. Exposure to cisplatin results also in an increased thermotolerance of EAT cells.

Supramolecular structure of the recombinant murine small heat shock protein hsp25.

The size and shape of the recombinant murine small heat shock protein, hsp25, have been analyzed by hydrodynamic and electron microscopic methods. According to these studies recombinant hsp25 exists in large complexes with a sphere-like shape and diameters of 15-18 nm. The molecular mass of these complexes amounts to about 730 kDa indicating that they are composed of about 32 monomers.

Identification of the phosphorylation sites of the murine small heat shock protein hsp25.

Native phosphorylated mouse small heat shock protein hsp25 from Ehrlich ascites tumor cells was isolated and the in vivo phosphorylation sites of the protein were determined. Furthermore, native hsp25 was phosphorylated by the endogenous kinase(s) in a cell-free system as well as recombinant hsp25 was phosphorylated in vitro by protein kinase C and catalytic subunit of cAMP-dependent protein kinase. The two major phosphorylation sites of native and recombinant hsp25 were determined as Ser-15 and Ser-86. There are no differences in the hsp25 phosphorylation sites phosphorylated by the protein kinase C, the catalytic subunit of cAMP-dependent protein kinase and the unknown intracellular kinase(s). The serine residues identified exist in all known small mammalian stress proteins and are located in the conserved kinase recognition sequence Arg-X-X-Ser.

Eukaryotic initiation factors eIF-2 and eIF-3: interactions, structure and localization in ribosomal initiation complexes.

More than ten different protein factors are involved in initiation of protein synthesis in eukaryotes. For binding of initiator tRNA and mRNA to the 40S ribosomal subunit, the initiation factors eIF-2 and eIF-3 are particularly important. They consist of several different subunits and form stable complexes with the 40S ribosomal subunit. The location of eIF-2 and eIF-3 in these complexes as well as the interactions of the individual components have been analyzed by biochemical methods and electron microscopy. The results obtained are summarized in this article, and a model is derived describing the spatial arrangement of eIF-2 and eIF-3 together with initiator tRNA and mRNA on the 40S subunit. Conclusions on the location of functionally important sites of eukaryotic small ribosomal subunits are discussed with regard to the respective location of these sites in the prokaryotic counterpart.

The 5'-untranslated region of p23 mRNA from the Ehrlich ascites tumor is involved in translation control of the growth related protein p23.

The growth-related protein p23 of the Ehrlich ascites tumor (EAT) is preferentially expressed in the exponentially growing tumor; its synthesis is translationally controlled. p23 mRNA is efficiently translated in the wheat germ cell-free lysate. In contrast, p23 mRNA present in poly(A)+RNA isolated from EAT is not translated in cell-free systems of EAT and reticulocytes. Moreover, translation of a p23 transcript is inhibited in the presence of total poly(A)+RNA. This inhibition is abolished by the removal of the 5'-UTR of the p23 transcript. Solution hybridization/RNase protection experiments point to the presence of a nucleotide sequence complementary to the 5'-UTR of p23 mRNA which might be involved in p23 mRNA inhibition.

Immunoelectron microscopic studies on the location of ribosomal proteins on the surface of the 40S ribosomal subunit from rat liver.

Seven ribosomal proteins have been localized by means of immunoelectron microscopy on the surface of the 40S ribosomal subunit from rat liver using monospecific antibodies. The location of ribosomal proteins S13/16, S19, and S24 is described for the first time, and that of ribosomal proteins S2, S3, S3a, and S7, which has been published previously on the basis of experiments performed with less well characterized antibody preparations [Lutsch et al., Mol. Gen. Genet. 176, 281-291 (1979) and Biomed. Biochim. Acta 42, 705-723 (1983)], is corrected in this paper. The results are discussed with respect to the involvement of these proteins in functional sites of the 40S ribosomal subunit.

The expression of the growth-related 25kDa protein (p25) of Ehrlich ascites tumor cells is increased by hyperthermic treatment (heat shock).

The abundance of a 25 kDa protein (p25) and at least two phosphorylated isoforms is inversely correlated with the rate of in vivo growth of the Ehrlich ascites tumor (EAT). On the basis of cDNA sequence data, p25 shows an about 80% amino acid sequence homology to the mammalian heat shock protein hsp27 (Gaestel et al., Europ. J. Biochem. 179, 209, 1989). In this paper, the following data are presented and discussed: 1. The expression of p25 is increased by in vitro incubation of cells at elevated temperatures (41.5 degrees C-43.5 degrees C). 2. A two-step hyperthermic treatment with a recovery period at 37 degrees C results in a more elevated p25 expression than a one-step hyperthermia. Moreover, the two-step hyperthermic treatment results in thermotolerance in terms of protein biosynthesis and cell vitality. 3. Also the appearance of p25 isoforms depends on the temperature regimes. While after a one-step hyperthermia the phosphorylated isoforms p25/2 and p25/3 predominate, the unphosphorylated isoform p25/1 is more strongly expressed after a two-step hyperthermic treatment, which results also in a more increased total p25 synthesis than a one-step treatment. 4. The synthesis of p25 and its induction by hyperthermic treatment in EAT cells depends on the stage of tumor growth from which the cells were harvested. Both, the endogenous synthesis and the induction by elevated temperature are higher in cells from the exponentially growing tumor than in cells from the stationary tumor. The results are discussed with respect to correlations between regulation of tumor growth and the abundance of p25, its synthesis and induction during tumor growth.

The growth-related protein P23 of the Ehrlich ascites tumor: translational control, cloning and primary structure.

p23 is a protein of Ehrlich ascites tumor cells, preferentially synthesized in the exponentially growing tumor. In vitro, serum and actinomycin D rapidly induce p23 synthesis. Using transcription inhibitors and a wheat germ cell-free translation system, evidence is provided that the synthesis of p23 is under translational control. Actinomycin D even results in superinduction of p23. Polymerase chain reaction, cloning and sequencing of p23 cDNA suggest p23 to be identical with a 21 kDa protein of mouse erythroleukemia cells, the synthesis of which was shown to be controlled also at the translational level (Chitpatima, S. T., Makrides, S., Bandyopadhyay, R., and Brawerman, G. (1988) Nucleic Acids Res. 16, 2350).

Structure of the rat liver ribosome 40 S subunit: freeze-drying and high-resolution shadow casting.

Small ribosomal subunits from rat liver have been studied by electron microscopy using freeze-drying and high-resolution shadow casting. The absolute hand of the asymmetric subunit has been determined and its three-dimensional model with a 'right' location of the side protuberance has been constructed. The results evidence that pro- and eukaryotic ribosomes have a unique and principally similar structural organization.

Purification of the growth-related protein p25 of the Ehrlich ascites tumor and analysis of its isoforms.

1. Two of the three isoforms of the growth-related protein p25 of the Ehrlich ascites tumor have been purified to homogeneity by giant two-dimensional polyacrylamide gel electrophoresis. 2. Antibodies raised against the isoform p25/1 react also with isoforms p25/2 and p25/3. 3. Limited tryptic digestion of p25/1 and p25/2 resulted in similar oligopeptide patterns. Corresponding oligopeptides of both isoforms have identical amino acid sequences. 4. The isoforms p25/2 and p25/3 are phosphorylated derivatives of unphosphorylated p25/1. The phosphorus is bound to serine and a further unknown phosphorylation site.

Identification of proteins of the 40 S ribosomal subunit involved in interaction with initiation factor eIF-2 in the quaternary initiation complex by means of monospecific antibodies.

Monospecific polyclonal antibodies against seven proteins of the 40 S subunit of rat liver ribosomes were used to identify ribosomal proteins involved in interaction with initiation factor eIF-2 in the quaternary initiation complex [eIF-2 X GMPPCP X [3H]Met-tRNAf X 40 S ribosomal subunit]. Dimeric immune complexes of 40 S subunits mediated by antibodies against ribosomal proteins S3a, S13/16, S19 and S24 were found to be unable to bind the ternary initiation complex [eIF-2 X GMPPCP X [3H]Met-tRNAf]. In contrast, 40 S dimers mediated by antibodies against proteins S2, S3 and S17 were found to bind the ternary complex. Therefore, from the ribosomal proteins tested, only proteins S3a, S13/16, S19 and S24 are concluded to be involved in eIF-2 binding to the 40 S subunit.

Shape and location of eukaryotic initiation factor eIF-2 on the 40S ribosomal subunit of rat liver. Immunoelectron-microscopic and hydrodynamic investigations.

The location of initiation factor eIF-2 and of its subunits in quaternary initiation complexes (40S-ribosomal-subunit.eIF-2. GuoPP[CH2]P.Met-tRNAf) was investigated by immunoelectron microscopy. Quaternary complexes were fixed with glutaraldehyde and reacted with affinity-purified polyclonal antibodies against eIF-2 alpha, eIF-2 beta or eIF-2 gamma. The dimeric immune complexes obtained by sucrose gradient centrifugation were investigated electron microscopically after negative staining. Antibody-binding sites were observed on the interface side of the 40S ribosomal subunit in the region between the 'head' and the 'body' (neck region) of the 40S ribosomal subunit. Within this region, eIF-2 alpha points to the rear side, whereas eIF-2 beta and eIF-2 gamma point to the frontal side of the 40S subunit indicating an elongated shape of eIF-2 about 15 nm long. By analytical ultracentrifugation of isolated eIF-2 the sedimentation and diffusion coefficients were determined to be 6.54 S and 4.74 x 10(-7) cm2/s respectively. From these data, a molar mass of 122.4 kg/mol and a dry volume of 147.4 nm3 were calculated. For the shape of eIF-2 a prolate ellipsoid of revolution is assumed with a maximal length of about 15 nm and with an axial ratio of about 1:3.5. This conclusion is further confirmed by a calculated frictional ratio of 1.37 and a Stokes radius of about 4.54 nm.